Long telomeres are preferentially extended during recombination-mediated telomere maintenance

Abstract

Most human somatic cells do not express telomerase. Consequently, with each cell division their telomeres progressively shorten until replicative senescence is induced. Around 15% of human cancers maintain their telomeres using telomerase-independent, recombination-based mechanisms that are collectively termed 'alternative lengthening of telomeres' (ALT). In the yeast Saccharomyces cerevisiae, ALT cells are referred to as 'survivors'. One type of survivor (type II) resembles human ALT cells in that both are defined by the amplification of telomeric repeats. We analyzed recombination-mediated telomere extension events at individual telomeres in telomerase-negative yeast during the formation of type II survivors and found that long telomeres were preferentially extended. Furthermore, senescent cells with long telomeres were more efficient at bypassing senescence by the type II pathway. We speculate that telomere length may be important in determining whether cancer cells use telomerase or ALT to bypass replicative senescence.

Figure 1

STEX analysis of type II survivor formation. (a) Senescence was monitored in liquid culture by serially passaging four est2Δ spore products derived from an est2Δ/+ heterozygous diploid. (b–d) Analysis of sequenced VI-R telomeres from three est2Δ strains at days 5, 6, and 7 – (b)est2Δ no. 1, (c) est2Δ no. 2, (d) est2Δ no. 3. Analysis of telomeres from est2Δ no. 4 is shown in Supplementary Figure 1. Each bar represents an individual VI-R telomere, which are sorted by the length of the undiverged sequence. The black portion of each bar represents the undiverged, or unextended, region of the telomere. The light grey portion of each bar represents the diverged, or extended, region of the telomere. For each est2Δ strain, the longest telomere without exhibiting divergent sequence, represented by a hashed bar, is used as a reference telomere to which all other telomeres are compared to determine if divergence has occurred.

Figure 2

Long telomeres are preferentially extended during type II survivor formation. (a) The sequenced VI-R telomeres from samples from pre-survivor cells (est2Δ no. 1, day 5; est2Δ no. 2, days 5 and 6; est2Δ no. 3, days 5 and 6; est2Δ no. 4, days 5 and 6) were pooled and analyzed together. A run of telomeres with a significant increase in telomeres that show divergence is indicated. The number of extended telomeres and total number of telomeres in the run is shown, as well as the p-value (determined by scan statistics; see Methods). (b) The sequenced telomeres from all samples where more than 30% but less than 70% of the telomeres show sequence divergence (est2Δ no. 1, day 6, telomere VI-R; est2Δ no. 3, day 7, telomere VI-R; est2Δ no. 4, day 7, telomeres VI-R and XIV-R) were pooled and analyzed together. A run of telomeres with a significant increase in telomeres that show divergence is indicated. The number of extended telomeres and total number of telomeres in the run is shown, as well as the p-value (determined by scan statistics; see Methods). (c) Long unextended telomeres from day 7 samples can be explained by one of two models, both involving the preferential, recombination-mediated extension of long telomeres. The top panel shows the est2Δ no. 3 data from Figure 1d. An orange line highlights the observation that a significant number of telomeres from the day 7 sample contains undiverged regions that are longer than the longest day 6 telomere. In Scenario A, the long telomeres from day 7 originated from a long telomere from day 6 (blue bar indicated by blue arrow) that was not sequenced because such long telomeres were too rare in the day 6 sample to be detected in our assay. In Scenario B, a long telomere from day 6 (red bar indicated by red arrow) was extended between days 6 and 7. This telomere was then duplicated several times, giving rise to the long telomeres in day 7. See text for more detail. Importantly, in both models, it is the long telomeres that are preferentially extended.

Figure 3

Telomerase-negative rifΔ strains exhibit accelerated senescence. (a) Telomere VI-R was sequenced from est2Δ rif1Δ, est2Δ rif2Δ, and two different isolates of est2Δ rif1Δ rif2Δ after ~35 generations of clonal expansion following sporulation of an est2Δ/+ rif1Δ/+ rif2Δ/+ diploid. Sequenced telomeres were analyzed as in Figure 1. (b) Senescence rates were measured in liquid culture by serial passaging of est2Δ, est2Δ rif1Δ, est2Δ rif2Δ, and est2Δ rif1Δ rif2Δ strains derived from the same diploid used in (a). The means and standard errors for at least four independent spore isolates for each genotype are shown.

Figure 4

Telomere shortening rate is unchanged when RIF1 and RIF2 are deleted. (a) One isolate each of est2Δ and est2Δ rif1Δ rif2Δ was passaged as in Figure 3b. (b) At each passage, telomere lengths were determined by denaturing in-gel hybridization (see Methods). The vertical bar to the right of the gel indicates the position of the terminal restriction fragments of Y’ telomeres, which are present in over half of yeast telomeres. The larger bands represent non-Y’-containing telomeres. A smear of telomeric signal appears in the est2Δ rif1Δ rif2Δ strain after 45 population doublings (PDs), which arise from type II survivors that have undergone telomere repeat amplification. (c) The telomere lengths from (b) were quantified and plotted.

Figure 5

Model for the senescence of est2Δ and est2Δ rif1Δ rif2Δ strains. In an est2Δ strain (on the left), telomeres progressively shorten with each cell division. Short telomeres are uncapped and recruit DNA damage factors like Rad52. The recruitment of DNA damage factors at one or a small number of shortened telomeres is insufficient to cause senescence until most or all telomeres have been sufficiently eroded. Bypass of senescence occurs either by the type I pathway (involving subtelomeric Y’ elements), or less frequently, the type II pathway (involving the telomeric repeats). The type II pathway involves the preferential extension of long telomeres by recombination. In an est2Δ rif1Δ rif2Δ strain (on the right), telomeres become uncapped earlier leading to accelerated senescence. However, since the telomere shortening rate is unaffected by deletion of the RIF genes, telomeres are relatively long at senescence as compared to telomeres in an est2Δ strain. The longer telomeres recombine more efficiently, resulting in a dramatic increase in the fraction of est2Δ rif1Δ rif2Δ survivors that are type II. The Rad52 protein complex is depicted as a hexamer for simplicity.